I'm trying to perform a histone blot, in my previous lab we used to add Benzonase to get rid of bound DNA and improve the blot quality, can DNAse I substitute Benzonase (since its expensive), and is it very necessary? Does someone have experience with running it without nucleases?
Thanks
Perhaps this protocol could help. It described different histone extraction methods, such as acid extraction and salt extraction which don't use DNAse I or Benzonase.
http://www.ncbi.nlm.nih.gov/pubmed/17545981
you can to add NUN buffer to your chromatin bounded pellet and to do sonication and then centrifugation ...thus in the supernatant your chromatin bounded proteins become " free" from DNA and suitable for runing on gel
see NUN buffer composition from WIKI http://en.wikipedia.org/wiki/NUN_buffer
NUN buffer resulting in a disruption of interaction between proteins and DNA. By incubating nuclei in NUN buffer and centrifuging the solution, the supernatant will therefore contain nuclear proteins., also do not forget to add PI Phi to your NUN buffer .
also this article is recommended http://www.nature.com/nprot/journal/v9/n9/full/nprot.2014.135.html
I tried adding DNAse to RIPA buffer for histone blot, it seemed to work fine, i didnt have any weird band, I used Antibodies from Active Motif. Thanks everyone for contribution
Cheap substitute for nonspecific nuclease is here: http://www.protean.cz/en/recombinant-protein/11/endonuclease-s-marcescens
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